We intend to study the mechanisms which render the nicotinic synapse a minute but highly sophisticated biophysical machine, specialized to function on a time scale of milliseconds and a distance scale of micrometers. Electrophysiological methods will be applied to three favorable and complementary preparations: Electrophorus electicus electroplaques, frog muscle fibers, and chick embryo myosacs. 1. We shall continue to measure the rate-limiting steps in the activation of acetylcholine receptor channels, with the goal of reconstructing the response to brief or sustained application of agonist. A) We shall compare the temporal information obtained from voltage-jump relaxations and from fluctuations of the conductance. B) Synaptic and extrasynaptic receptors will be compared with respect to their opening and closing rates. C) We shall exploit a new technique, the photochemically produced agonist concentration-jump. 2. We shall study the molecular nature of these rate-limiting steps. On a millisecond time scale, binding of fluorescent drugs (agonists, antagonists and local anesthetics) will be measured and compared with the simultaneously measured membrane conductance. 3. With micro-iontophoretically applied agonists and antagonists, we shall study how drug diffusion is altered by multiple binding to receptors in the synaptic cleft. These measurements will provide estimates of physiologically significant binding affinities. 4. We shall study the functional significance of voltage-sensitive channels in Electrophorus electroplaques. Action potentials will be measured when voltage-insensitive shunts have been introduced into the membrane or the external solutions.